Use of MALDI-MS for the analysis of small molecules, such as pharmacologically active constituents and also metabolites, is only partially possible since these molecules fall into a mass range, which usually cannot be concerned with this analysis technique. This fact is caused by the used matrix, which is normally necessary to analyse intact molecules (usually peptides, proteins). Minimum size of the analytes should therefore be 500 to 700μ, in the ideal case greater than 1000μ.
Standard systems for the screening of small molecules are gas chromatography and liquid chromatography coupled to mass spectrometry (GC-MS, LC-MS). An essential disadvantage of these systems lies in long and time consuming sample preparation steps and GC/LC-MS runs of 20 min or longer, which restricts daily throughput of samples. In contrast, MALDI-MS can reach a high automated throughput.
In the literature several examples of systems which allow a direct analysis of small molecules by MALDI-MS can be found. Within these examples a stainless steel target is used and modified so far that laser energy of 337 nm can be absorbed. This is the case for porous silicon layers (Zhang et al. Rap. Commun. Mass Spect. 2001 15 217-223; Shen et al. Anal. Chem. 2001 73 612-619; Jing Wei et al. Nature 1999 399 243-246; Kraj et al. Acta Biochimica Polonica 2003 50 (3) 783-787), but can be reached also by use of polymers (Peterson et al. Rap. Commun. Mass Spect. 2004 18 1504-1512; Frechet et al. U.S. 20050023456 A1) or by use of special modified surfaces, such as silica modified with triphenylmethane or silica modified with matrix systems like α-cyano-hydroxy cinnamomic acid (HCCA) or 2,5-dihydroxybenzoic acid (DHB) (Zhang et al. Rap. Commun. Mass Spect. 2001 15 217-223).
Immobilised carbon nanotubes are a further possibility (Shi-fang Ren et al. JASMS 2005 16 (3) 333-339) next to graphite (Hie-Joon Kim et al. Anal. Chemical 2000 72 5673-5678) or a combination of sample with inorganic particles, such as Mn, Mo, Si, Sn, TiO2, W, WO3, Zn, ZnO (Kinumi et al. J. Mass Spectr. 2000 35 417-422). Fonash et al. disclose in their patent application the use of amorphous silicon-layers and porous SiO2-layers for the matrix free MALDI-MS (Fonash et al. WO 02/093170 A1, US20020144456). Hutchens describes the use of azodianiline for the immobilisation of biomolecules by means of photolabile attachment (Ching et al. J. Org. Chem. 1996 61 3582-3583; Hutchens et al. U.S. Pat. No. 6,124,137).
Within this last cited patent a clear division between photo-labile attachment and matrix free MALDI-MS is given: For matrix free MALDI-MS immobilized matrices such as HCCA or DHB were used, whereas for photo-labile attachment azodianiline is described.
The object of the present invention is therefore to provide a system which allows to use MALDI-MS for the high throughput screening of molecules having a molecular weight lower than 700μ (lowest measured mass is sodium with m/z=23, FIG. 15), e.g. for pharmacologically active compounds as well as for drug metabolites and for secondary plant metabolites. Additionally, the system should be suitable also for target compounds in the range up to several 1000μ. As most of described and published systems show dominant background signals, a further object is the establishment of a system without interfering signals or with a limited number of background signals.